Developments in biomedical research and material science rely increasingly on state-of-the-art instruments capable of structural imaging and chemical analysis at very high spatial resolution.
Far-field imaging approaches are usually diffraction limited. In the far field, chemical imaging of small features, such as nanoparticles or nanostructures which fall to a size range below 100 nm, requires thus a breakaway from the diffraction limit. A number of techniques providing chemical imaging at nanoscale resolution have been developed. However, the resolution is then either achieved using near-field techniques or in the far-field by using very short wavelengths (e.g., X-ray, electron microscopy).
In addition to nanoparticles and nanostructures, the nanometer scale is also typical of biological molecules involved in photosynthesis, color-control, and biochemical reactivity. Intracellular analysis in living cells, and the study of large biomolecules generally ranging from 10 to 200 nm, are also of interest.
Progress in these R&D fields requires the development of microscope(s) enabling the chemical characterization of materials with spatial resolution of the order of 1 to 500 nm.
The limited ability to routinely probe and understand the properties of matter at sub-cellular and at nanometer scale hinders progresses and new tools and methodologies need thus to be conceptualized and their effectiveness demonstrated.
Direct measurement of vibrational absorption by optical means requires the use of infrared (IR) beam (wavelength ranges approximately between 750 nm and 1 mm). However, contrary to ultraviolet (UV) and/or to fluorescent microscopy, which uses relatively smaller wavelengths of light (about 10-750 nm), resolutions below the micrometer range seems impossible to achieve in far-field IR microscopy, in view of the limits as expressed by the Ernest Abbe criterion, which forbids spatial resolution better than approximately half of the wavelength of the probe beam.
Chemical bonds in a molecule vibrate at a characteristic frequency. A group of atoms in a molecule may have multiple modes of oscillation. If an oscillation leads to a change in dipole in the molecule, then it will absorb a photon which has the same frequency. The vibrational frequencies of most molecules occur within the infrared light frequency ranges. Because vibrational modes are dependent on composition and on local molecular arrangement, they serve as a fingerprint of molecules, and mapping of the spatial distribution of these modes provides a mean of label-free imaging without the need for any chemically binding additives (labels). This mapping of vibrational signatures is also called chemical imaging, spectroscopic imaging or spectro-microscopy. Current state-of-the-art far-field microscopes affording a mapping of vibrational modes based on CARS (coherent anti-Stoke Raman spectroscopy), vibrational SFG (sum-frequency generation), SRS (stimulated Raman microscopy), or on IRAS (infrared absorption spectroscopy) exhibit a spatial resolution that is at best limited by diffraction.
For microscopy in Fourier Transform Infra Red Absorption Spectroscopy (FT-IRAS) mode using an IR synchrotron source the best resolution that are achieved are then limited to several microns only.
Other instrumental FT-IRAS setup using thermal sources are unable to reach these values due the poor brightness of these sources and generally the resolution is limited to about 20 microns.
Today to achieve higher resolution in IRAS it is necessary to exploit near-field scanning optical microscopies (NSOM), which require to maintain a nanoscale solid probe in the vicinity of the sample and which are thus limited to probing the surface of samples, and exhibit a large technical difficulty due to the poor reliability of probe production and to the necessity to maintain it at nanometer range from the sample. These techniques generally afford spatial resolution of the order of 100 nm.
Although NSOM afford extremely high spatial resolution in IRAS, it seems thus important to consider new techniques to achieve comparable or better resolution in the far-field, which will suppress the limitation to surface-only probing and the engineering challenges related to the nanoscale probe exploitation. However in this case, one needs to find a scheme to achieve in IRAS resolution that overcomes the diffraction limit.
The measurement of the chemical IR absorption with a resolution below the diffraction limit also cannot be done using the techniques developed for sub-diffraction far-field imaging of fluorescence emission. These relies on the controlled suppression of the fluorescence emission (e.g., STED stimulated-emission depletion), or on the localization of randomly activated fluorescent chromophores (e.g., PALM photo-activated localization microscopy, STORM stochastic optical reconstruction microscopy), or on the analysis of Moiré patterns of the emitted fluorescence (e.g., SSIM saturated structured illumination microscopy). These methods of fluorescence imaging are also not label-free since they necessitate the incorporation of fluorescent tag/label in the sample.
Since IRAS probes the intrinsic vibrational modes of molecules, it is thus label-free and it is thus extremely important to find scheme in the far-field that will afford below-the-diffraction-limit resolution for IRAS.